【摘要】 目的构建晚期糖化终产物受体(RAGE)反义RNA腺相关病毒载体,并在大鼠肾脏系膜细胞中表达。方法构建腺相关病毒介导的RAGE反义RNA载体,3个质粒共转染293细胞,获得病毒原液,感染大鼠肾脏系膜细胞,流式细胞术、RT-PCR、ELISA检测重组病毒感染的细胞RAGE的表达和分泌细胞外基质的情况。结果经酶切鉴定、序列分析显示RAGE基因片段正确完整反向插入pAAV-MCS。利用293细胞包装获得病毒原液的滴度为8.7×107VP/mL。感染重组病毒的细胞与正常细胞比较RAGE表达被抑制(48.2±6.1)%,分泌Ⅳ型胶原(ColⅣ)水平明显下降(P<0.05)。结论成功构建具有抑制功能的RAGE反义RNA腺相关病毒载体,为进一步研究RAGE的作用机制,以及基因治疗RAGE相关疾病提供一个重要工具。更多还原

【Abstract】 Objective To construct recombinant receptor for advanced glycation endproducts (RAGE)antisense RNA adeno-associated virus vector and express the vector in rat mesangial cell. Methods Total RNA was acquired by TRIZOL method and the gene fragment of rat RAGE was amplified by RT-PCR. The target gene was connected to pAAV-MCS vector, and then identified by restriction endonuclease digestion and DNA sequence analysis. Recombinant AAV-RAGE- was derived by co-transfecting DNAs of rAAV-RAGE-, pAAV-RC, and pAAV-helper into AAV-293 cells using liposome methods. The titer of recombinant AAV-RAGE- was calculated by staining the cells expressed the report gene LacZ with X-Gal. After transduction of recombinant AAV-RAGE- in rat mesangial cell, flow cytometry (FCM) and RT-PCR were used to determine the expression of RAGE and ELISA was used to examine the levels of fibronectin (FN) and type Ⅳ collagen (ColⅣ) in cell supernatant induced by AGEs. Results The recombinant AAV-RAGE- was obtained with the titers of 8.7×107 virus particles/mL. After AGEs induction, the expressions of RAGE was inhibited by (48.2±6.1)% and the amount of ColⅣ in the medium was lower than that of vector virus transducted cells (P<0.05). Conclusion The success in constructing recombinant AAV/RAGE- promotes the further research in molecular biology of RAGE and the recombinant AAV/RAGE- can be potentially used in gene therapy for RAGE associated diseases.更多还原